There has been known a method for assaying an endotoxin with the use of limulus amebocyte lysate (hereinafter referred to simply as lysate) by taking advantage of a fact that said lysate coagulates with a trace amount of an endotoxin. Subsequent biochemical studies have revealed that this coagulation is caused by stepwise activation of several coagulation factors [cf. Takanori Nakamura et al., Japanese Journal of Bacteriology, 38,781-803 (1983)].
When an endotoxin is added to the lysate, as shown in FIG. 1, factor C (an endotoxin-sensitive factor, molecular weight: 123,000) is activated. Then, the activated factor C definitively hydrolyzes and activates factor B (molecular weight: 64,000). The activated factor B thus formed activates proclotting enzyme (molecular weight: 54,000) and thus convert the same into clotting enzyme. The clotting enzyme definitively hydrolyzes specific sites, i.e., Arg.sup.18 -Thr.sup.19 and Arg.sup.46 -Gly.sup.47, of coagulogen (a coagulation protein, molecular weight: 19,723) and thus liberates peptide C to thereby convert the coagulogen into coagulin, which causes coagulation (gelation). Iwanaga et al. [Haemostasis, Z, 183-199 (1978)] further proposed a highly quantitative assay method wherein the lysate is combined with a synthetic peptide having an amino acid sequence in common with the above-mentioned hydrolysis sites of coagulogen, namely a chromogenic substrate Boc-Leu-Gly-Arg-p-nitroanilide (pNA) or a fluorogenic substrate Boc-Leu-Gly-Arg-4-methylcoumaryl-7-amide.
This assay method depends on a series of reactions involving a cascade mechanism wherein an endotoxin acts as a trigger so as to successively activate coagulation factors all of which are serine protease precursors and thus the coagulin is formed finally.
When (1 .fwdarw. 3)-.beta.-D-glucan is added to the lysate, factor G shown in FIG. 1 is activated and the activated factor G thus formed converts the proclotting enzyme into the clotting enzyme. Next, the clotting enzyme converts the coagulogen into the coagulin, similar to the case of the endotoxin, to thereby form a gel and hydrolyzes a synthetic substrate [cf. Morita et al., FEBS Lett., 129, 318-321 (1981)].
Known examples of substances reacting with factor G include (1 .fwdarw. 3)-.beta.-D-glucan, krestin, lentinan and substances contained in rinses from cellulosic hemodialyzer and blood contacted therewith. It has been confirmed that none of these substances would show any positive reaction in a rabbit pyrogenic test.
Endotoxin is also known as cell wall components of gram-negative bacteria. Thus, the presence of gram-negative bacteria in vivo can be detected by determining endotoxin in blood. Accordingly, it has been urgently required in the field of clinical diagnosis to establish a method of assaying an endotoxin at a high sensitivity and a high reproducibility without being affected by (1 .fwdarw. 3)-.beta.-D-glucan.
Obayashi et al. reported a method of assaying an endotoxin by using factor C-system in the lysate [cf. Clin. Chim. Acta, 149, 55-65 (1985)]. However, this method requires a highly complicated procedure since it comprises fractionating the lysate by gel filtration or affinity chromatography with the use of a heparin or dextran sulfate-immobilized carrier, eliminating factor G sensitive to (1 .fwdarw. 3)-.beta.-D-glucan and thus reconstituting the lysate with factors C and B and the proclotting enzyme.